Specific expression of FABP5 in type II alveolar epithelial cells in silicosis pulmonary fibrosis based on single-cell transcriptome sequencing
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摘要:背景
矽肺是由于长期吸入大量游离的二氧化硅（SiO2）颗粒所致，探讨其机制可以为矽肺纤维化的治疗提供新的方向。
目的本研究着重探讨脂肪酸结合蛋白5（FABP5）在SiO2诱导的矽肺模型中的表达及发挥的作用。
方法结合前期单细胞转录组测序结果，利用生物信息分析技术对小鼠肺泡上皮细胞进行分群并探索FABP5的表达模式；采用空间转录组学技术探索
fabp5 的分布模式。小鼠气管滴注SiO2建立矽肺纤维化模型，设置4个组别：生理盐水（normal saline, NS）7 d组、NS 56 d 组、SiO2 7 d组、SiO2 56 d组。SiO2处理小鼠肺上皮细胞系（MLE-12）建立矽肺体外模型。在动物整体水平，利用组织免疫荧光实验检测上皮细胞的标志物（E-Cad）和FABP5的蛋白水平；在体外水平，利用MLE-12验证细胞模型中fabp5 mRNA 表达变化和蛋白水平变化。结果单细胞转录组测序结果及空间转录组测序结果显示在小鼠矽肺病灶区域II型肺泡上皮细胞中
fabp5 mRNA表达上调，伴随着组织免疫荧光蛋白水平升高，且与E-Cad存在共定位现象。同时SiO2刺激诱导MLE-12细胞中fabp5 mRNA表达升高至1.58倍和蛋白水平上升至2倍，差异均具有统计学意义（P <0.05）。结论在肺纤维化模型的肺泡上皮细胞中，FABP5的蛋白水平上升，提示FABP5可能参与上皮细胞在肺纤维化病理进程。
Abstract:BackgroundSilicosis is caused by long-term inhalation of large amounts of free silica (SiO2) particles, and exploring its mechanism can provide new directions for the treatment of silicosis fibrosis.
ObjectiveTo investigate the expression and role of fatty acid binding protein 5 (FABP5) in a silica-induced silicosis model.
MethodsIn combination with the results of single-cell transcriptome sequencing, the expression pattern of FABP5 in mouse alveolar epithelial cells was explored by bioinformatic analysis, and the distribution pattern of
fabp5 was detected by spatial transcriptomics. Anin vivo model of silicosis was established by intratracheal injection with SiO2 into mice and four groups were set up: normal saline (NS) 7 d group, NS 56 d group, SiO2 7 d group, and SiO2 56 d group. Anin vitro model of silicosis was established in SiO2-treated mouse lung epithelial cell line (MLE-12). At the whole animal level, the marker of epithelial cells (E-Cad) and the protein level of FABP5 were detected by tissue immunofluorescence assay;in vitro , the changes offabp5 mRNA expression and protein level in MLE-12.ResultsThe results of single-cell transcriptome sequencing and spatial transcriptome sequencing showed that the mRNA expression of
fabp5 was upregulated in type II alveolar epithelial cells in the focal area of silicosis in mice, accompanied by elevated tissue immunofluorescent protein levels, and there was co-localization of E-CAD. Meanwhile, SiO2 stimulation induced a 1.58-fold increase infabp5 mRNA expression and a 2-fold increase in protein levels in MLE-12 cells, with significant differences (P <0.05).ConclusionThe protein level of FABP5 is increased in alveolar epithelial cells in a pulmonary fibrosis model, suggesting that FABP5 may be involved in the pathological process of epithelial cells in pulmonary fibrosis.
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图 1 矽肺单细胞转录组测序标志基因气泡图展示
Figure 1. Bubble diagram display of silicosis single cell transcriptome sequencing marker genes
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图 2 单细胞转录组测序中fabp5细胞总群中的小提琴图展示
Figure 2. Violin diagram display of fabp5 cell population in single cell transcriptome sequencing
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图 3 AT2细胞亚型中fabp5不同表达模式的小提琴图展示
Figure 3. Violin diagram display of different expression patterns of fabp5 in AT2 cell subtypes
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图 5 fabp5表达阳性的AT2细胞所占全部AT2细胞的比例
Figure 5. The proportion of fabp5-positive AT2 cells in total AT2 cells
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图 6 fabp5阳性表达的AT2细胞区域和肺纤维化病灶区
上：HE染色，圈出部分为病灶区域；下：空间转录组学fabp5阳性表达的AT2细胞区域，圈出区域为阳性表达区域。
Figure 6. fabp5 positive expression in AT2 cell regions and pulmonary fibrosis lesions
Upper: HE staining, circle indicates lesions; Lower: Spatial transcriptome sequencing, circle indicates fabp5 positive expression in AT2 cells.
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图 7 SiO2刺激MLE-12细胞不同时间后构建的离体模型中qPCR检测fabp5的mRNA表达
*：与0 h相比， P<0.05。
Figure 7. qPCR detection results of mRNA expression of fabp5 in the in vitro model of MLE-12 cells stimulated by SiO2 over time
*: Compared with 0 h, P<0.05.
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图 8 SiO2刺激MLE-12细胞不同时间后构建的离体模型中蛋白免疫印记法检测FABP5的蛋白水平
*：与0 h相比，P<0.05。
Figure 8. Western blotting detection results of the protein levels of FABP5 in the in vitro model of MLE-12 cells stimulated by SiO2 over time
*: Compared with 0 h, P<0.05.
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图 9 肺组织FABP5蛋白水平的免疫荧光显示
Figure 9. Immunofluorescence of FABP5 protein levels in lung tissues
表 1 目的基因及内参基因的引物序列
Table 1 Primer sequence of target gene and internal reference gene
基因(Gene) 正向引物(5′—3′) 反向引物(5′—3′) fabp5 CACGGCTTTGAGGAGTACATC AGGTGCAGACCGTCTCAGTTT gapdh ACCATCTTCCAGGAGCGAGTA GGGCAGAGATGATGACCCTTT 
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[1] 《2019年我国卫生健康事业发展统计公报》发布[J]. 职业卫生与应急救援, 2020, 38(3): 214. Statistical bulletin on the development of China's health undertakings in 2019[J]. Occup Health Emerg Rescue, 2020, 38(3): 214.
[2] PRAKASH J, PINZANI M. Fibroblasts and extracellular matrix: targeting and therapeutic tools in fibrosis and cancer[J]. Adv Drug Deliv Rev, 2017, 121: 1-2. doi: 10.1016/j.addr.2017.11.008
[3] HEWLETT J C, KROPSKI J A, BLACKWELL T S. Idiopathic pulmonary fibrosis: epithelial-mesenchymal interactions and emerging therapeutic targets[J]. Matrix Biol, 2018, 71-72: 112-127. doi: 10.1016/j.matbio.2018.03.021
[4] 宫欢欢, 许伟, 祝红, 等. 基于中西医临床病症特点的矽肺动物模型分析[J/OL]. 中药药理与临床, 1-12. (2022-07-20)[2022-07-25]. http://dx.doi.org/10.13412/j.cnki.zyyl.20220719.005. GONG H H, XU W, ZHU H, et al. Animal models of silicosis based on clinical symptoms in Chinese and western medicine[J/OL]. Pharmacol Clin Chin Mater Med, 1-12. (2022-07-20)[2022-07-25]. http://dx.doi.org/10.13412/j.cnki.zyyl.20220719.005.
[5] ADAMS T S, SCHUPP J C, POLI S, et al. Single-cell RNA-seq reveals ectopic and aberrant lung-resident cell populations in idiopathic pulmonary fibrosis[J]. Sci Adv, 2020, 6(28): eaba1983. doi: 10.1126/sciadv.aba1983
[6] RAO D M, PHAN D T, CHOO M J, et al. Impact of fatty acid binding protein 5-deficiency on COPD exacerbations and cigarette smoke-induced inflammatory response to bacterial infection[J]. Clin Transl Med, 2019, 8(1): e7.
[7] OHATA T, YOKOO H, KAMIYAMA T, et al. Fatty acid-binding protein 5 function in hepatocellular carcinoma through induction of epithelial-mesenchymal transition[J]. Cancer Med, 2017, 6(5): 1049-1061. doi: 10.1002/cam4.1020
[8] O'SULLIVAN S E, KACZOCHA M. FABP5 as a novel molecular target in prostate cancer[J]. Drug Discov Today, 2020, 25(11): 2056-2061. doi: 10.1016/j.drudis.2020.09.018
[9] LIU X, FANG S, LIU H, et al. Role of human pulmonary fibroblast-derived MCP-1 in cell activation and migration in experimental silicosis[J]. Toxicol Appl Pharmacol, 2015, 288(2): 152-160. doi: 10.1016/j.taap.2015.07.002
[10] QI Y, ZHAO A, YANG P, et al. miR-34a-5p Attenuates EMT through targeting SMAD4 in silica-induced pulmonary fibrosis[J]. J Cell Mol Med, 2020, 24(20): 12219-12224. doi: 10.1111/jcmm.15853
[11] LEVI L, LOBO G, DOUD M K, et al. Genetic ablation of the fatty acid-binding protein FABP5 suppresses HER2-induced mammary tumorigenesis[J]. Cancer Res, 2013, 73(15): 4770-4780. doi: 10.1158/0008-5472.CAN-13-0384
[12] LEE D, WADA K, TANIGUCHI Y, et al. Expression of fatty acid binding protein 4 is involved in the cell growth of oral squamous cell carcinoma[J]. Oncol Rep, 2014, 31(3): 1116-1120. doi: 10.3892/or.2014.2975
[13] XIE T, WANG Y, DENG N, et al. Single-cell deconvolution of fibroblast heterogeneity in mouse pulmonary fibrosis[J]. Cell Rep, 2018, 22(13): 3625-3640. doi: 10.1016/j.celrep.2018.03.010
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